PROJECT SUMMARY/ABSTRACT Ubiquitin-mediated proteolysis regulates the degradation of numerous proteins, thereby controlling many cellular processes, including cell cycle progression, signal transduction pathways, differentiation, and the centrosome duplication cycle. Much of the specificity inherent in the ubiquitination process is mediated by the E3 ubiquitin ligases, which bind selectively to, and recruit, the chosen substrate to the ubiquitin-conjugating enzyme. Notably, the majority of ubiquitin ligases are considered ?orphan?, because their substrates have not yet been identified. Several ubiquitin ligases localize to the centrosome and control the ubiquitination and subsequent proteasomal degradation of critical centriole duplication factors, such as CP110, PLK4, and SAS6 (by SCF-Cyclin F, SCF-?TrCP, and APC/C-Cdh1, respectively). Furthermore, ubiquitin ligases also control additional centrosomal functions, such as centriole separation (through the SCF-?TrCP-mediated degradation of Cep68). Our preliminary results show that additional ?orphan? E3 ligase complexes reside at the centrosome, and that several centrosomal proteins are degraded by the ubiquitin system during specific phases of the cell cycle. We propose a project systematically exploring the regulation of the centrosome cycle by the ubiquitin-proteasome system. We will use proteomic techniques to identify novel substrates of centrosomal E3 ligases (specific AIM 1) and will validate and biochemically characterize the most biologically significant substrates identified under AIM 1 (specific AIM 2). We will make our analysis of E3 ubiquitin ligases and centrosome interactors available as a web-accessible resource. Centrosome amplification is a common feature of the large majority of cancers and can result in chromosome instability. Furthermore, centrosome abnormalities are also associated with genetic disorders of neurons and cellular cilia. Therefore, the information gained from the proposed studies is expected to be of direct relevance to our understanding of cancer biology and other human diseases such as ciliopathies and neuronal development disorders.